Oct4 is required ~E7.5 for proliferation in the primitive streak.

Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

Abstract

Oct4 is a widely recognized pluripotency factor as it maintains Embryonic Stem (ES) cells in a pluripotent state, and, in vivo, prevents the inner cell mass (ICM) in murine embryos from differentiating into trophectoderm. However, its function in somatic tissue after this developmental stage is not well characterized. Using a tamoxifen-inducible Cre recombinase and floxed alleles of Oct4, we investigated the effect of depleting Oct4 in mouse embryos between the pre-streak and headfold stages, ~E6.0-E8.0, when Oct4 is found in dynamic patterns throughout the embryonic compartment of the mouse egg cylinder. We found that depletion of Oct4 ~E7.5 resulted in a severe phenotype, comprised of craniorachischisis, random heart tube orientation, failed turning, defective somitogenesis and posterior truncation. Unlike in ES cells, depletion of the pluripotency factors Sox2 and Oct4 after E7.0 does not phenocopy, suggesting that ~E7.5 Oct4 is required within a network that is altered relative to the pluripotency network. Oct4 is not required in extraembryonic tissue for these processes, but is required to maintain cell viability in the embryo and normal proliferation within the primitive streak. Impaired expansion of the primitive streak occurs coincident with Oct4 depletion ∼E7.5 and precedes deficient convergent extension which contributes to several aspects of the phenotype.

A The fraction (Oct4f/f;CreERT2+/−/total) embryos recovered at each developmental stage (‘@’ indicates resorbing embryos). Embryos were administered tamoxifen ∼E7.0 and ∼E7.5 (). B,C Representative images of an E10.5 Oct4f/f;CreERT2+/− embryo and Oct4f/f littermate following tamoxifen administration ∼E7.0 and ∼E7.5. Features such as the otic cup (∧) and forelimb bud (#) which normally arises after NTC is initiated and turning is complete are present in both Oct4f/f;CreERT2+/− and Oct4f/f littermates. Conversely, defects in turning, somitogenesis, neural tube closure and posterior extension all fail to recover in Oct4f/f;CreERT2+/− embryos by E10.5. Scale bars in ‘B,C’ are 250 µm. B Sagittal view. C Dorsal view. D Schematic illustrating NT closure points, the directions in which the NT ‘zippers’ shut as well as the sections where mesenchyme density was quantified. E,F Representative Hematoxylin and Eosin (H and E) sections from the trunk (see ‘Tr’ in panel ‘D’) ∼E9.5. Differences in the density of mesenchyme, which is indicated with two-headed arrows, are apparent. Embryos were administered tamoxifen ∼E7.0 and ∼E7.5 and dissected ∼E9.5 (). Scale bars in E,F are 50 µm. E Oct4f/fF Oct4f/f;CreERT2+/−. G Mesenchyme, connective tissue comprised of mesoderm and neural crest cells, has a lower density in Oct4COND MUT. The average density within 4×10−2 mm2 ±s.e.m. (200 µm×200 µm) from sections equivalent to those in panel ‘E,F’ is plotted (F1,13 = 54.60, p<0.05 2-way ANOVA, *p<0.05, ***p<0.001 Bonferroni posttest). Due to the posterior truncation, insufficient mesenchyme was present in the tail of Oct4COND MUT to quantify. H–J The neural tube in H Oct4f/fI Oct4f/f;CreERT2+/−. Scale bars in ‘H,I’ are 100 µm. J The distance between neural folds in the trunk of Oct4f/f;CreERT2+/− is broader (F2,22 = 17.42, p<0.05 2-way ANOVA, **p<0.01 Bonferroni posttest. Embryos in ‘H–J’ were administered tamoxifen ∼E7.0 and ∼E7.5 and dissected ∼E9.5 ().

Tetraploid chimeras indicate that Oct4 is not required extraembryonically ∼E7.5, while diploid chimeras indicate that Oct4+/+ can compensate for Oct4−/− cells embryonically.

All embryos transferred to a surrogate and depicted or described in panels A–E were induced with tamoxifen ∼E6.0 and ∼E6.5 to compensate for the variability in developmental timing associated with transfer (,AB). Scale bars in panels A–D are 200 um. A A representative embryo from aggregation of Oct4+/+ RFP ES cells with a tetraploid Oct4f/f embryo. Oct4f/f extraembryonic tissue yielded E9.5 chimeric embryos with no phenotype. B An Oct4f/f;CreERT2+/− E9.5 embryo with the Oct4COND MUT phenotype. C Oct4 depletion in extraembryonic tissue is compatible with WT development. A representative chimera consisting of RFP+ Oct4+/+ ES cell derived embryo and tetraploid Oct4f/f;CreERT2+/− extraembryonic tissue. The embryo has turned (compare panel ‘B’ where the tail is behind to panel ‘C’ where it is in front), undergone NTC and posterior extension (compare the lack of somites and short tail in panel ‘B’ to the somites and full-length tail in ‘C’). D The most severe embryonic defect observed in a diploid chimeras consisting of Oct4+/+ and Oct4f/f;CreERT2+/− cells. The neural tube is open between closure points 1 and 2, indicated here with a black bracket. All Oct4COND MUT features aside from cranial NTC defect, which is still present in 5/16 mosaic embryos, are rescued by Oct4+/+ cells in these diploid chimeras (16/16). For example, this embryo has ‘turned’ such that it faces its tail and the posterior has extended normally. E Quantification of the genotypes and phenotypes of recovered chimeric embryos.

A–C Sox2f/f;CreERT2+/− embryos were administered tamoxifen ∼E6.5 and ∼E7.0 and dissected ∼E9.5 (D). A Sagittal view of Sox2f/f;CreERT2+/− and Sox2f/f littermates. 11/20 Sox2f/f;CreERT2+/− embryos had hydrocephalus ∼E9.5. B Dorsal view of a Sox2f/f;CreERT2+/− embryo with hydrocephalus. Two-headed arrows indicate region where neuroepithelium does not approach the midline of Sox2f/f;CreERT2+/− as it does in Sox2f/f embryos. C 2/20 Sox2f/f;CreERT2+/− had kinked neural tubes (the kinking is indicated with arrows) without hydrocephalus (at left and right), while the embryo in the middle has the more prevalent hydrocephalus. Neither hydrocephalus nor kinked neural tubes were observed in Oct4COND MUT embryos.

Pathway enrichment and confirmation of a subset of differentially expressed genes following Oct4 depletion.

Litters depicted in ‘A,B’ were induced with tamoxifen ∼E7.0 (E). The FDR for reported enrichments in ‘A,B’ is <0.001, based on 1000 random permutations of annotated genes. A Unsupervised clustering of relative (Oct4f/f;CreERT2+/−/Oct4f/f littermates) gene expression sub-setted for Oct4 binding targets following Oct4 depletion. Enrichment for the same pathways in the global differential expression set and subset directly targeted by Oct4 support the utility of sub-setting for Oct4 binding targets in identifying primary effects of Oct4 depletion and the relevance of these primary effects to the Oct4COND MUT phenotype in that they appear amplified into effects on the overall gene expression profile (see blue script in panel ‘A’ and ‘B’ for these). The most enriched pathway is provided for each cluster, and an additional pathway provided (in black text) for the cluster where the most enriched pathway in the Oct4 target set did not translate to a global change. B Unsupervised clustering of global differential expression (same dataset as panel ‘A’): Oct4f/f;CreERT2+/−/Oct4f/f. The most enriched pathway and binding factor are provided for each cluster (black text), while primary effects that translated to enriched effects in the global set are in blue text. C Confirmation of expression change for select genes by quantitative PCR in independent litters (Oct4f/f;CreERT2+/−/Oct4f/f ±s.e.m.). Litters were induced with tamoxifen ∼E7.0 (G).